1. The Field of the Invention
This invention relates to a method of identifying sequence alterations in a DNA fragment. More particularly the invention relates to a solution-based method of scanning for single base pair substitutions using fluorescence melting profiles and a double-stranded DNA binding dye.
2. Technical Background
Many human diseases and disorders are associated with genetic alterations. Many diseases can be attributed to a change or a mutation in a single gene. The diseases causes by genetic mutations may be diagnosed based on detection of a mutation within the genome of an individual. With early detection and the proper diagnosis, many of these disease may be treated. However, many of the currently available methods of detection are costly and labor intensive. Because of the labor involved the methods are subject to human error and run the risk of false results.
Several types of mutations can occur in DNA. A point mutation occurs when a single base is changed to one of the three other bases. A deletion occurs where one or more bases are deleted from a gene. An insertion occurs where new bases are inserted at a particular point in a nucleic acid sequence adding additional length to the sequence.
Large insertions or deletions can be readily detected within a DNA sample. Because of the small degree of molecular change, the point mutation is the most difficult type of mutation to screen for and detect. However, a number of diseases including some types of cancer are known to be caused by point mutations.
Methods that permit the detection of single base changes in specific regions of the genome have enjoyed tremendous utility in the field of genetics by facilitating genetic linkage analysis and the identification of mutations with specific disease associations, including those involved in the development and evolution of neoplasia. R. Wallace et al., Science 249:181-6 (1990); R. M. Cawthon et al., Cell 62:193-201 (1990); R. A. Flavell et al., Cell 15:25-41 (1978); A. P. Feinberg et al., Science 220:1175-1177 (1983); J. L. Bos et al., Nature 327:293-297 (1987); Hollstein et al., Science 253:49-53 (1991).
Single base alterations have been detected using a variety of methods. Some methods rely on the abolition or creation of novel restriction enzyme sites for example, restriction fragment length polymorphism analysis. Other methods use the polymerase chain reaction (PCR) and subsequent distinction of base mismatches by oligonucleotide hybridization. Single base changes have also been detected by the differences in the conformational or melting temperature characteristics of the mutated and wild-type sequences for example, single strand conformation polymorphism analysis and denaturation gradient gel electrophoresis. A. R. Wyman and R. White, Proc Natl Acad Sci USA 77:6754-6758 (1980); K. Mullis et al., Cold Spring Harb Symp Quant Biol 51:263-273 (1986); R. K. Saiki et al., Science 239:487-91 (1988); A. Neri et al., Proc Natl Acad Sci USA 85:9268-9272 (1988); M. Orita et al., Proc Natl Acad Sci USA 86:2766-70 (1989). All of these methods require multiple steps including gel electrophoretic separation and/or radioisotopic detection of the sequence variants. The use of radioisotopes creates safety concerns that increase the cost and limit the utility of these methods.
Recently, fluorescence-based technologies have been used for the detection of specific nucleic acid sequences. R. Higuchi et al., Biotechnology (N Y) 11:1026-30 (1993). These assays have typically exploited one of several fluorescence chemistries. C. T. Wittwer et al., Biotechniques 22:130-8 (1997). Some of these assays are non-specific methods and incorporate a double stranded DNA (dsDNA) binding dye such as SYBR(copyright) Green I into the amplification reaction. The specificity of product detection is entirely dependent on the inherent specificity of the amplification conditions. Subtle changes such as point mutations are difficult to detect using non-specific dsDNA binding dyes.
Other fluorescence-based assays are sequence-specific. These methods are probe-based and incorporate oligonucleotides that hybridize to a sequence within the amplified sample sequence. This method provides an additional parameter for verification of product identity. The specificity of the hybridization interaction has been further exploited for the identification of single nucleotide polymorphisms by virtue of the fact that the single base mismatches within the hybridization probe to DNA target hybrids exhibit lower melting temperatures than perfectly complementary strands. However, the detection of single base changes using probe-based methods is limited to very short segments of DNA of approximately 20 base pairs or less. The probe-based methods would require several probes in multiple separate reactions to scan a region of more than 100 bases. The cost of such probe-based assays is relatively high. Thus, the probe-based methods are unsuitable for mutational scanning of larger regions of DNA.
Unstacking of long helical DNA fragments has been shown in denaturing gel-based systems to occur in a succession of segments or discrete cooperative units referred to as domains. The melting temperatures (Tms) of these domains are principally determined by the base composition and precise DNA sequence. Single base differences in otherwise homologous DNA fragments can be discriminated provided that the differences are located within the lowest melting domain, and this domain is clearly separated from the onset of duplex to single strands dissociation. Both of these conditions can be satisfactorily achieved by the attachment of a higher melting section or a GC-rich sequence to the DNA fragment of interest, thus rendering the detection of virtually all single base changes possible. These principles have been exploited in such gel-based approaches as denaturation gradient gel electrophoresis and related methods. However, the gel-based systems require a significant amount of time, and are not readily adaptable to automation.
In light of the foregoing, it would be a significant advancement in the art to provide a solution-based method for detecting changes in a DNA sequence capable of screening DNA segments of greater than 20 base pairs. It would be an additional advancement if the method were capable of distinguishing even subtle single base changes from the wild-type. It would be an additional advancement if the method used a double-stranded DNA binding dye for detecting the mutation. It would be an additional advancement if the method were rapid and not labor intensive. It would be a further advancement if the method produced accurate and reproducible results. It would be an additional advancement if the method were adaptable to automation.
Such a method is disclosed herein.
The present invention relates to a solution-based method for determining whether a DNA sequence is identical to a wild-type sequence. The alteration may be a point mutation such as a transversion or transition or other mutation such as an insertion or deletion. The method uses a dsDNA binding dye and fluorescent melting profiles to detect a mutation within a DNA segment of interest.
In a presently preferred embodiment, a sample of DNA suspected of containing a mutation is obtained. The sequence to be analyzed for the presence of an alteration is amplified to obtain an adequate amount of the sequence. A GC-rich segment is attached to the 5xe2x80x2 end of the sequence of interest. This GC-clamp creates two melting domains, a higher domain associated with the GC-clamp and a lower domain associated with the DNA segment of interest. The GC-clamp may be of any length or sequence that confers a significantly higher melting temperature than that estimated for the sequence of interest.
In a presently preferred embodiment of the invention, a double stranded DNA (dsDNA) binding dye such as SYBR(copyright) Green I (Molecular Probes, Eugene, Oreg.), ethidium bromide, or YO-PRO-I(copyright) (Molecular Probes, Eugene, Oreg.) is included in an amplification reaction. The dsDNA binding dye attaches to the DNA as double-stranded amplicons are formed. The dye will continue to bind the DNA segment so long as the DNA remains double-stranded. Thus, the denaturation of the dsDNA will be observed as a significant reduction in fluorescence.
In one presently preferred embodiment of the invention, a sequence alteration may be detected by slowly heating the fluorescently labeled, GC-clamped DNA amplicon in the presence of a constant concentration of a denaturant. The mixture of the DNA segment of interest and denaturant is heated from below the melting temperature of the DNA segment of interest to above the melting temperature of the GC-clamp. In a presently preferred embodiment, the mixture is heated from about 33xc2x0 C. to about 95xc2x0 C. In a presently preferred embodiment of the invention, the mixture is heated at a rate less than 0.5xc2x0 C./second. Preferably, the mixture is heated at a ramp rate of between about 0.01xc2x0 C./second and about 0.1xc2x0 C./second. More preferably, the rate of heating is about 0.02xc2x0 C./second.
The fluorescence of the reaction mixture is monitored to determine the melting temperature of the segment of interest. Because the fluorescent label dissociates from the DNA as the DNA is melted, the melting point is seen as a significant reduction in fluorescence. In this manner, a mutation may be detected by comparing the melting point of a test DNA segment of interest to the melting point of the wild-type sequence. A difference in melting temperatures may indicate a mutation in the test DNA segment of interest.
The denaturant may be selected from a variety of denaturants such as urea. It is presently preferred that urea be used at a concentration of about 13.3M.
When a DNA sample contains a homozygous alteration in a sequence of interest, the melting temperature of the DNA sample, may be indistinguishable from that of a sample containing only wild-type sequence. This is especially true for point mutations such as transitions where the thermodynamic stability of the mutant and the wild-type sequence differ only very slightly. The method of the present invention may be adapted to detect such a mutation.
In a presently preferred embodiment, a polymorphism may be detected by creating an artificial heterozygote. This is accomplished by obtaining a sample of the DNA segment of interest. An approximately equal amount of the wild-type sequence is mixed with the sample sequence. The dsDNA dye generated melting temperature of the sample DNA:wild-type heteroduplex may then be compared to the melting temperature of the wild-type DNA to determine if the sample DNA contains one or more alterations.
The mixture is then amplified by PCR. The cyclical denaturation and renaturation of jointly amplifying the DNA sample and the wild-type sequence with PCR creates heteroduplexes. The heteroduplex is then mixed with a denaturant and slowly heated as described above. As the heteroduplex melts, the dsDNA dye dissociates from the DNA and a reduction in fluorescence is observed. The heteroduplex will exhibit two melting peaks in the lower melting domain or a broad transition in the lower melting domain. This is attributable to the presence of wild-type:wild-type, mutant:mutant, and mutant:wild-type duplexes in the reaction mixture. Often, the preparation of an artificial heterozygote is not necessary as the mutation that is being screened for is heterozygous in its natural state.
In summary, a solution-based method for determining whether a DNA sequence is identical to a wild-type sequence comprises coupling a GC-rich DNA segment to a DNA sample containing at least one copy of the DNA segment. The DNA sample in question is subjected to thermal cycling amplification which includes a dsDNA binding dye, and the reaction products are mixed with a denaturant and heated while monitoring fluorescence to determine the melting point of the amplicon. Germline heterozygous mutations form heteroduplexes with wild-type sequences, and this complex exhibits a lower melting temperature than wild-type homoduplexes. Homozygous mutations may be detected by combining approximately equal parts of the DNA sample with approximately equal parts of the wild-type sequence to create a heteroduplex. The double-stranded DNA bound heteroduplex is mixed with a denaturant and heated. A difference in melting temperatures of the DNA sample or the heteroduplex and the wild-type sequence indicates an alteration in the DNA sequence. This solution-based method is capable of reproducibly screening DNA segments greater than about 20 base pairs for alterations as small as a single base change. In the present embodiment, the region of interrogation is the entire length of a 104 bp DNA segment. The method is relatively rapid, requiring only about 40 minutes of post-amplification analysis, and is adaptable to automation.
These and other advantages of the present invention will become apparent upon reference to the accompanying drawings and graphs and upon reading the following detailed to description and appended claims.